DETERMINATION OF THE ASSOCIATION OF MYOSIN SUBFRAGMENT-1 WITH ACTIN IN THE PRESENCE OF ATP

The mechanism of the actomyosin ATPase is multistep involving cyclic attachment and detachment of myosin and F-actin. The fraction of myosin bound to actin in the presence of substrate gives information about the distribution of myosin between these states. We have determined the fraction of rabbit myosin subfragment 1 (S-l) bound to actin in the presence of ATP, using an ultracentrifuge to separate bound and free S-l. Estimates have also been made of the extent of binding from turbidity measurements. These experiments were carried out at a number of different actin concentrations, both at 0.5 and 20 °C and at 17 and 35 mM salt concentrations. The fraction cf S-1 bound at a given actin concentration was compared with the percentage saturation of the actin-activated ATPase at the same actin concentration, in an attempt to determine whether the rate-limiting step in the ATPase mechanism occurs when the S-l is bound to actin or involves a transition of S-l in the unbound state. At 20 °C, there was a reasonably good correlation between the fraction of S-l bound to actin and the percentage saturation of the actoS-1 ATPase, while at 0.5 °C the fraction bound was much less than the saturation of the actoS-1 ATPase. These results suggest that the rate-con- trolling processes are different at 20 and 0.5°C. Another approach to the problem is to compare the maximal rate of ATP hydrolysis per mole of S-l at infinite actin concentration with that expressed per mole of actin at infinite S-l concentration. Under the latter conditions, one would expect the maximal turnover of ATP to be determined by the rate of product release from the actin-S-1 -ADP-Pi complex, whereas, under the former, any slow step involving S-l alone would become apparent. Our experiments showed that at 5°C the rate of ATP hydrolysis per mole of actin was two to four times faster than that per mole of S-l, but at 20 °C the difference between these rates was only 30%, again suggesting differences in the rate-controlling processes as a function temperature. The results of the binding measurements and steady-state ATPase activities indicate that at 20 °C the rate of product release from the actin-S- 1-ADP-P;i complex is similar to, if not the same as, the steady-state rate in the actoS-1 ATPase; thus, a large fraction of S-l may exist in this form at high actin concentrations. At low temperatures, only a small fraction of the S-l is complexed with actin even at very high actin concentrations. © 1979, American Chemical Society. All rights reserved.